Identifying target genes of the aryl hydrocarbon receptor nuclear translocator (Arnt) using DNA microarray analysis

The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic helix-loop-helix (bHLH) protein that also contains a Per-Arnt-Sim (PAS) domain. In addition to forming heterodimers with many other bHLH-PAS proteins, including the aryl hydrocarbon receptor (AhR) and hypoxia-inducible factors 1alpha, 2alpha and 3alpha, Arnt can also form homodimers when expressed from its cDNA in vitro or in vivo. However, target genes of the Arnt/Arnt homodimer remain to be identified. In this study, we have elucidated the profile of genes responsive to the reintroduction of Arnt expression in an Arnt-deficient mouse hepatoma cell line (c4), using DNA microarray analysis. The expression of 27 genes was upregulated by 1.5-fold or more in c4 cells infected with a retroviral vector expressing mouse Arnt, while no genes were found to be downregulated. Among the upregulated genes, BCL2/adenovirus E1B 19 kDa-interacting protein 1 (NIP3), serine (or cysteine) proteinase inhibitor, clade E, member 1 (PAI1), and N-myc downstream regulated-like (NDR1), were confirmed to be induced by Arnt using real-time PCR. We also found that the 5' promoter region of 15 out of 20 upregulated genes contain the type 2 E-box 5'-CACGTG-3' Arnt/Arnt binding sequence, consistent with the notion that they represent target genes for Arnt.     

Microarray analyses of hypoxia-regulated genes in an aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent manner

We investigated hypoxia-inducible factor (HIF)-dependent changes in the expression of 5592 genes in response to hypoxia (0.1% O(2), 16 h) by performing cDNA microarray analyses of mouse hepa1c1c7 and BpRc1 cells. BpRc1 cells are a hepa1c1c7 variant defective in HIF-beta/aryl hydrocarbon receptor nuclear translocator (Arnt), and are therefore unable to induce HIF target genes in response to hypoxia. By comparing hepa1c1c7 cells with BpRc1 cells, we were able to investigate hypoxia-regulated gene expression as well as the role played by HIF in regulating the hypoxic-dependent response of gene expression. This study identified 50 hypoxia-induced genes and 36 hypoxia-repressed genes. Quantitative PCR analysis of nine genes confirmed our ability to accurately analyze changes in hypoxia-induced gene expression by microarray analysis. By comparing quantitative PCR analyses of these nine genes in BpRc1 and hepa1c1c7 cells, we determined that eight of the nine hypoxia-induced genes are Arnt dependent. Additional quantitative PCR analyses of eight hypoxia-repressed genes confirmed, with a 50% probability, that microarray analysis was able to predict hypoxia-repressed gene expression. Only two of the four confirmed genes were found to be repressed in an Arnt-dependent manner. Collectively, six of these 13 genes (46.2% probability) showed a pattern of expression consistent with the microarray analysis with regard to Arnt dependence. Finally, we investigated the HIF-1alpha dependence of these 13 genes by quantitative PCR analysis in HIF-1alpha knockdown 3T3-L1 cells. These analyses identified novel hypoxia-regulated genes and confirmed the role of Arnt and HIF-1alpha in regulating their expression. These results identify additional HIF target genes and provide a more complete understanding of hypoxia signaling.     

芳香烃受体核转位蛋白的结构及相关功能

芳香烃受体核转位蛋白（aryl hydrocarbon receptor nuclear translocator,ARNT）是碱性螺旋-环．螺旋转录因子超家族中新发现的PAS亚家族的成员之一。它是体内许多bHLH-PAS蛋白共同的专性配偶体,可以与芳香烃受体、低氧诱导因子、果蝇SIM蛋白等形成异二聚体并介导许多信号转导过程,从而使个体对环境污染物（如二恶英）、低氧状态等外界因素的改变产生相应的生物学效应,本文就ARNT的基本结构及其在体内的主要生理功能等方面作一综述。     

Conditional disruption of the aryl hydrocarbon receptor nuclear translocator (Arnt) gene leads to loss of target gene induction by the aryl hydrocarbon receptor and hypoxia-inducible factor 1alpha

To determine the function of the aryl hydrocarbon receptor nuclear translocator (ARNT), a conditional gene knockout mouse was made using the Cre-loxP system. Exon 6, encoding the conserved basic-helix-loop-helix domain of the protein, was flanked by loxP sites and introduced into the Arnt gene by standard gene disruption techniques using embryonic stem cells. Mice homozygous for the floxed allele were viable and had no readily observable phenotype. The Mx1-Cre transgene, in which Cre is under control of the interferon-gamma promoter, was introduced into the Arnt-floxed mouse line. Treatment with polyinosinic-polycytidylic acid to induce expression of Cre resulted in complete disruption of the Arnt gene and loss of ARNT messenger RNA (mRNA) expression in liver. To determine the role of ARNT in gene control in the intact animal mouse liver, expression of target genes under control of an ARNT dimerization partner, the aryl hydrocarbon receptor (AHR), was monitored. Induction of CYP1A1, CYP1A2, and UGT1*06 mRNAs by the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin was absent in livers of Arnt-floxed/Mx1-Cre mice treated with polyinosinic-polycytidylic. These data demonstrate that ARNT is required for AHR function in the intact animal. Partial deletion of the Arnt allele was found in kidney, heart, intestine, and lung. Despite more than 80% loss of the ARNT expression in lung, maximal induction of CYP1A1 was found, indicating that the expression level of ARNT is not limiting to AHR signaling. Cobalt chloride induction of the glucose transporter-1 and heme oxygenase-1 mRNAs was also markedly abrogated in mice lacking ARNT expression, suggesting an inhibition of HIF-1alpha activity. These studies establish a critical role for ARNT in AHR and HIF-1alpha signal transduction in the intact mouse.     

Regulatory factors involved in species-specific modulation of arylhydrocarbon receptor (AhR)-dependent gene expression in humans and mice

The arylhydrocarbon receptor (AhR) mediates toxicities of dioxins, including the most potent congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), by being translocated to the nucleus upon ligand-binding and inducing expression of target genes. Although the species-specific activity of the AhR is primarily attributable to species-specific AhR-ligand affinity, the precise mechanism has not been clarified. We investigated the modulation mechanisms of AhR in Hepa1c1c7 and HepG2 hepatoma cells, which were derived from high-affinity-AhR-expressing C57BL/6 mice and low-affinity-AhR-expressing humans, respectively. Although, consistent with their AhR affinities, TCDD induced a greater amount of cytochrome P450 1A1 (CYP1A1) mRNA, one of the most sensitive AhR-targets, in Hepa1c1c7 cells than in HepG2 cells immediately after exposure, both cells expressed a similar level of CYP1A1 mRNA from 4 h onward. A rapid decrease in the AhR protein after nuclear translocation in Hepa1c1c7 cells was suggested to contribute to suppression of CYP1A1 induction to the same level as in HepG2 cells. Different profiles of histone deacetylase 1 (HDAC1)-binding to the CYP1A1 promoter and histone acetylation between both cell lines and lower degradation rate of CYP1A1 mRNA in HepG2 cells were also implicated in regulating their target gene expression. These factors have been highly suggested to be involved in the species-specific modulation mechanism of AhR function.     

Proteomic dissection of the von Hippel-Lindau (VHL) interactome

The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex containing elongin B, elongin C, cullin 2, and Rbx1, which acts as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the alpha subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in the maintenance of primary cilium, extracellular matrix formation, and tumor suppression. We undertook a series of proteomic analyses to gain a comprehensive picture of the VHL-interacting proteins. We found that the ARF tumor suppressor interacts with VHL30, a longer VHL isoform, but not with VHL19, a shorter VHL isoform. ARF was found to release VHL30 from the E3 ligase complex, promoting the binding of VHL30 to a protein arginine methyltransferase, PRMT3. Our analysis of the VHL19 interactome also uncovered that VHL19 displays an affinity to collagens and their biosynthesis enzymes.     

Genotype-phenotype correlations in families with deletions in the von Hippel-Lindau (VHL) gene

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome characterized by predisposition for bilateral and multi-centric hemangioblastoma in the retina and central nervous system, pheochromocytoma, renal cell carcinoma, and cysts in the kidney, pancreas, and epididymis. We describe five families for which direct sequencing of the coding region of the VHL gene had failed to identify the family-specific mutation. Further molecular analysis revealed deletions involving the VHL gene in each of these families. In four families, partial deletions of one or more exons were detected by Southern blot analysis. In the fifth family, FISH analysis demonstrated the deletion of the entire VHL gene. Our results show that (quantitative) Southern blot analysis is a sensitive method for detecting germline deletions of the VHL gene and should be implemented in routine DNA diagnosis for VHL disease. Our data support the previously established observation that families with a germline deletion have a low risk for pheochromocytoma. Further unraveling of genotype-phenotype correlations in VHL disease has revealed that families with a full or partial deletion of the VHL gene exhibit a phenotype with a preponderance of central nervous system hemangioblastoma.     

Inactivation of oestrogen receptor in vitro by nuclear dephosphorylation.

1. Nuclei of the calf uterus are endowed with an activity inactivating crude oestrogen-receptor complex. This activity has been partially purified. It shows a very high affinity for the oestrogen-receptor complex (Km = 0.8 X 10(-9) mol of specific [3H]oestradiol-17 beta-binding sites/l) as well as for the oestrogen-free receptor (Km = 1.5 X 10(-9) mol of specific [3H]oestradiol-17 beta binding sites/l). 2. The nuclear receptor-inactivating activity is enhanced by dithiothreitol and inhibited by several phosphatase inhibitors as well as by 4-nitrophenyl phosphate, as well known phosphatase substrate. This inhibition shows that a dephosphorylation process is required for the receptor inactivation. 3. The purified nuclear activity also inactivates pure receptor and phosphatase inhibitors prevent this inactivation. From these observations it appears that receptor inactivation is due to a nuclear phosphatase directly acting on the oestrogen receptor. 4. The nuclear localization of the receptor-inactivating activity, its high affinity for specific oestrogen binding sites and, as previously reported, its presence only in oestrogen target tissues suggest that this activity is the same as that involved in the nuclear loss of the receptor observed in intact cells.     

Impact of the arylhydrocarbon receptor on eugenol- and isoeugenol-induced cell cycle arrest in human immortalized keratinocytes (HaCaT)

Fragrances such as eugenol (4-allyl-2-methoxyphenol) and isoeugenol (2-methoxy-4-propenylphenol), naturally found in reasonable quantities in the essential oils of different spices, are not only common causes of contact dermatitis but also known for their antiproliferative actions. Previously, we found a cell cycle arrest and an arylhydrocarbon receptor (AhR)-mediated activation of cytochromes in immortalized keratinocytes (HaCaT) induced by both compounds. In the present study we investigated whether the cell cycle arrest of eugenol and isoeugenol is mediated by the AhR in HaCaT cells. Analysis of the cell cycle status by fluorescence-activated cell sorting (FACS) revealed an arrest of cells (32-34%) in the G0/G1 phase induced by both compounds. This was found in synchronized HaCaT cells, natural HaCaT, and siRNA AhR transfected HaCaT. The induced G0/G1 arrests were reduced in the presence of the highly selective AhR antagonist 3'-methoxy-4'-nitroflavone (MNF). In summary, these results, together with our previous findings that both compounds induce translocation of the AhR into the nucleus, provide good evidence that the effects of eugenol and isoeugenol in skin and keratinocytes are mediated by the AhR. Furthermore, these data suggest that the known growth suppressive effects of these compounds in some skin cells may be mediated by AhR interactions.     

Adding structural information to the von Hippel-Lindau (VHL) tumor suppressor interaction network

The von Hippel-Lindau (VHL) tumor suppressor gene is a protein interaction hub, controlling numerous genes implicated in tumor progression. Here we focus on structural aspects of protein interactions for a list of 35 experimentally verified protein VHL (pVHL) interactors. Using structural information and computational analysis we have located three distinct interaction interfaces (A, B, and C). Interface B is the most versatile, recognizing a refined linear motif present in 17 otherwise non-related proteins. It has been possible to distinguish compatible and exclusive interactions by relating pVHL function to interaction interfaces and subcellular localization. A novel hypothesis is presented regarding the possible function of the N-terminus as an inhibitor of pVHL function.     

Expression of the mediators of dioxin toxicity,aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT), is developmentally regulated in mouse teeth

Dioxins are persistent and ubiquitous environmental poisons that become enriched in the food chain. Besides being acutely lethal, the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to many animal species. We have previously found that developing teeth of children may be sensitive to environmental dioxins via their mother's milk and that rat and mouse teeth are dioxin-sensitive throughout their development. The aryl hydrocarbon receptor (AHR) together with the AHR nuclear translocator (ARNT) protein is believed to mediate the toxic effects of dioxins. To study the potential involvement of the AHR-ARNT pathway in the dental toxicity of TCDD, we analysed the expression of AHR and ARNT by in situ hybridization and immunohistochemistry in developing mouse teeth. AHR mRNA first appeared in the epithelium of E12 first molar tooth buds and both proteins were weakly expressed in the bud. After cytodifferentiation the expression was up regulated and became intense in secretory odontoblasts and ameloblasts. The coexpression of AHR and ARNT during early tooth development as well as during the information and mineralization of the dental matrices is suggestive of the AHR-ARNT pathway as a mediator of dental toxicity of TCDD.